Transcriptome Analysis Reveals Enhancement of Cardiogenesis-Related Signaling Pathways by S-Nitroso- N -Pivaloyl- d -Penicillamine: Implications for Improved Diastolic Function and Cardiac Performance.

ABSTRACT: We previously reported a novel compound called S-nitroso- N -pivaloyl- d -penicillamine (SNPiP), which was screened from a group of nitric oxide donor compounds with a basic chemical structure of S-nitroso- N -acetylpenicillamine, to activate the nonneuronal acetylcholine system. SNPiP-treated mice exhibited improved cardiac output and enhanced diastolic function, without an increase in heart rate. The nonneuronal acetylcholine-activating effects included increased resilience to ischemia, modulation of energy metabolism preference, and activation of angiogenesis. Here, we performed transcriptome analysis of SNPiP-treated mice ventricles to elucidate how SNPiP exerts beneficial effects on cardiac function. A time-course study (24 and 48 hours after SNPiP administration) revealed that SNPiP initially induced Wnt and cyclic guanosine monophosphate-protein kinase G signaling pathways, along with upregulation of genes involved in cardiac muscle tissue development and oxytocin signaling pathway. We also observed enrichment of glycolysis-related genes in response to SNPiP treatment, resulting in a metabolic shift from oxidative phosphorylation to glycolysis, which was suggested by reduced cardiac glucose contents while maintaining adenosine tri-phosphate levels. In addition, SNPiP significantly upregulated atrial natriuretic peptide and sarcolipin, which play crucial roles in calcium handling and cardiac performance. These findings suggest that SNPiP may have therapeutic potential based on the pleiotropic mechanisms elucidated in this study.

The reactions of hydropersulfides (RSSH) with myoglobin.

Hydropersulfides are reported to be good biological reductants, superior to thiols and akin to selenols. As such, they have been previously shown to reduce metalloproteins such as ferric myoglobin and ferric cytochrome c to their ferrous forms under conditions where little or no reduction from corresponding thiols is observed. Not surprisingly, the reduction of ferric myoglobin to ferrous myoglobin under aerobic conditions results in the generation of oxymyoglobin (dioxygen bound ferrous myoglobin). Previous studies have demonstrated that oxymyoglobin can also act as an oxidant with highly reducing species such as hydroxylamine and ascorbate. Considering the reducing properties of hydropersulfides, it is possible that they can also react with oxymyoglobin similarly to hydroxylamine or ascorbate. Herein, this reaction is examined and indeed hydropersulfides are found to react with oxymyoglobin similarly to other reducing species leading to a fleeting ferric myoglobin which is rapidly reduced to the ferrous form also by hydropersulfide.

Cysteine/Penicillamine Ligation Independent of Terminal Steric Demands for Chemical Protein Synthesis.

The chemical ligation of two unprotected peptides to generate a natural peptidic linkage specifically at the C- and N-termini is a desirable goal in chemical protein synthesis but is challenging because it demands high reactivity and selectivity (chemo-, regio-, and stereoselectivity). We report an operationally simple and highly effective chemical peptide ligation involving the ligation of peptides with C-terminal salicylaldehyde esters to peptides with N-terminal cysteine/penicillamine. The notable features of this method include its tolerance of steric hinderance from the side groups on either ligating terminus, thereby allowing flexible disconnection at sites that are otherwise difficult to functionalize. In addition, this method can be expanded to selective desulfurization and one-pot ligation-desulfurization reactions. The effectiveness of this method was demonstrated by the synthesis of VISTA (216-311), PD-1 (192-288) and Eglin C.

S-Nitroso-N-Pivaloyl-D-Penicillamine, a novel non-neuronal ACh system activator, modulates cardiac diastolic function to increase cardiac performance under pathophysiological conditions.

We have previously reported the development of a novel chemical compound, S-Nitroso-N-Pivaloyl-D-Penicillamine (SNPiP), for the upregulation of the non-neuronal cardiac cholinergic system (NNCCS), a cardiac acetylcholine (ACh) synthesis system, which is different from the vagus nerve releasing of ACh as a neurotransmitter. However, it remains unclear how SNPiP could influence cardiac function positively, and whether SNPiP could improve cardiac function under various pathological conditions. SNPiP-injected control mice demonstrated a gradual upregulation in diastolic function without changes in heart rate. In contrast to some parameters in cardiac function that were influenced by SNPiP 24 h or 48 h after a single intraperitoneal (IP) injection, 72 h later, end-systolic pressure, cardiac output, end-diastolic volume, stroke volume, and ejection fraction increased. IP SNPiP injection also improved impaired cardiac function, which is a characteristic feature of the db/db heart, in a delayed fashion, including diastolic and systolic function, following either several consecutive injections or a single injection. SNPiP, a novel NNCCS activator, could be applied as a therapeutic agent for the upregulation of NNCCS and as a unique tool for modulating cardiac function via improvement in diastolic function.

Nitric oxide-releasing vascular grafts: A therapeutic strategy to promote angiogenic activity and endothelium regeneration.

Small-diameter vascular grafts (SDVGs) are associated with a high incidence of failure due to infection and obstruction. Although several vascular grafts are commercially available, specific anatomical differences of defect sites require patient-based design and fabrication. Design and fabrication of such custom-tailored grafts are possible with 3d-printing technology. The aim of this study is to develop 3d-printed SDVGs with a nitric oxide (NO)-releasing coating to improve the success rate of implantation. The SDVGs were printed from polylactic acid and coated with blending of 10 wt% S-nitroso-N-acetyl-D-penicillamine into the polymeric substrate consisting of poly (ethylene glycol) and polycaprolactone. Our results show that NO is released in the physiological range (0.5-4 × 10-10 mol·cm-2·min-1) for 14 days and NO-releasing coating showed significant antibacterial potential against Gram-positive and Gram-negative strains. It was shown that both NO-releasing and control grafts are biocompatible in-vitro and in-vivo. Interestingly, the NO-releasing SDVGs dramatically enhanced ECs proliferation and significantly enhanced ECs migration in-vitro compared to control grafts. In addition, the NO-releasing SDVGs showed angiogenic potential in-vivo which can further prove the results of our in-vitro study. These findings are expected to facilitate tissue regeneration and integration of custom-made vascular implants with enhanced clinical success. STATEMENT OF SIGNIFICANCE: A series of 3d-printed small-diameter vascular grafts (SDVGs, <6 mm) with controlled release of nitric oxide (NO) were prepared to combine the advantages of 3D printing technology and NO-releasing systems. The resulting NO-releasing grafts were promisingly showing sustained NO release in the physiological range over a two weeks period. In addition to the evaluation of endothelial cell migration in-vitro, we implanted for the first time the NO-releasing vascular grafts in a chick chorioallantoic membrane (CAM) to investigate the effect of the prepared grafts on the angiogenesis in-vivo. The fabricated grafts also exhibited bactericidal properties which prevent the formation of a biofilm layer and can thereby enhance the chance of endothelialization on the surface. Taken together, the innovative combination of rapid and highly accurate 3d-printing technology as a patient-specific fabrication method with NO-releasing coating represents a promising approach to develop bactericidal SDVGs with improved endothelialization.

Stimulation of constitutive nitric oxide uniquely and compensatorily regulates intestinal epithelial cell brush border membrane Na absorption.

In the mammalian small intestine, sodium is primarily absorbed by Na+ /H+ exchange (NHE3) and Na-glucose cotransport (SGLT1) in the brush border membrane (BBM) of villus cells. However, how enhanced cellular constitutive nitric oxide (cNO) may affect NHE3 and SGLT1 remains unclear. Both in vivo in rabbit intestinal villus cells and in vitro IEC-18 cells, administration of NO donor, GSNAP, modestly increased cNO. GSNAP stimulated SGLT1 in villus and IEC-18 cells. The mechanism of stimulation was secondary to an increase in the affinity of SGLT1 for glucose. The change in SGLT1 was not secondary to altered Na-extruding capacity of the cell since Na+ /K+ -ATPase was decreased by GSNAP treatment. In contrast, GSNAP inhibited NHE3 activity in villus cell BBM. The mechanism of NHE3 inhibition was secondary to reduced BBM transporter numbers. These studies demonstrated that the physiological increase in cNO uniquely regulates mammalian small intestinal NHE3 and SGLT1 to maintain Na homeostasis.

Achieving Long-Term Biocompatible Silicone via Covalently Immobilized S-Nitroso- N-acetylpenicillamine (SNAP) That Exhibits 4 Months of Sustained Nitric Oxide Release.

Ever since the role of endogenous nitric oxide (NO) in controlling a wide variety of biological functions was discovered approximately three decades back, multiple NO-releasing polymeric materials have been developed. However, most of these materials are typically short lived due to the inefficient incorporation of the NO donor molecules within the polymer matrix. In the present study, S-nitroso- N-acetyl penicillamine (SNAP) is covalently attached to poly(dimethylsiloxane) (PDMS) to create a highly stable nitric oxide (NO) releasing material for biomedical applications. By tethering SNAP to the cross-linker of PDMS, the NO donor is unable to leach into the surrounding environment. This is the first time that a sustainable NO release and bacterial inhibition for over 125 days has been achieved by any NO-releasing polymer with supporting evidence of potential long-term hemocompatibility and biocompatibility. The material proves to have very high antibacterial efficacy against Staphylococcus aureus by demonstrating a 99.99% reduction in the first 3 days in a continuous flow CDC bioreactor, whereas a similar inhibitory potential of 99.50% was maintained by the end of 1 month. Hemocompatibility of SNAP-PDMS was tested using a rabbit extracorporeal circuit (ECC) model over a 4 h period. Thrombus formation was greatly reduced within the SNAP-PDMS-coated ECCs compared to the control circuits, observing a 78% reduction in overall thrombus mass accumulation. These results demonstrate the potential of utilizing this material for blood and tissue contacting biomedical devices in long-term clinical applications where infection and unwanted clotting are major issues.

Effects of Mild Blast Traumatic Brain Injury on Cerebral Vascular, Histopathological, and Behavioral Outcomes in Rats.

To determine the effects of mild blast-induced traumatic brain injury (bTBI), several groups of rats were subjected to blast injury or sham injury in a compressed air-driven shock tube. The effects of bTBI on relative cerebral perfusion (laser Doppler flowmetry [LDF]), and mean arterial blood pressure (MAP) cerebral vascular resistance were measured for 2 h post-bTBI. Dilator responses to reduced intravascular pressure were measured in isolated middle cerebral arterial (MCA) segments, ex vivo, 30 and 60 min post-bTBI. Neuronal injury was assessed (Fluoro-Jade C [FJC]) 24 and 48 h post-bTBI. Neurological outcomes (beam balance and walking tests) and working memory (Morris water maze [MWM]) were assessed 2 weeks post-bTBI. Because impact TBI (i.e., non-blast TBI) is often associated with reduced cerebral perfusion and impaired cerebrovascular function in part because of the generation of reactive oxygen and nitrogen species such as peroxynitrite (ONOO-), the effects of the administration of the ONOO- scavenger, penicillamine methyl ester (PenME), on cerebral perfusion and cerebral vascular resistance were measured for 2 h post-bTBI. Mild bTBI resulted in reduced relative cerebral perfusion and MCA dilator responses to reduced intravascular pressure, increases in cerebral vascular resistance and in the numbers of FJC-positive cells in the brain, and significantly impaired working memory. PenME administration resulted in significant reductions in cerebral vascular resistance and a trend toward increased cerebral perfusion, suggesting that ONOO- may contribute to blast-induced cerebral vascular dysfunction.

Liquid-infused nitric oxide-releasing (LINORel) silicone for decreased fouling, thrombosis, and infection of medical devices.

Recent reports on liquid-infused materials have shown promise in creating ultra-low fouling surfaces, but are limited in their ability to prevent bacterial proliferation and prevent platelet activation in blood-contacting applications. In this work, a liquid-infused nitric oxide-releasing (LINORel) material is created by incorporating the nitric oxide (NO) donor S-nitroso-acetylpenicillamine (SNAP) and silicone oil in commercial medical grade silicone rubber tubing through a solvent swelling process. This combination provides several key advantages over previous NO-releasing materials, including decreased leaching of NO donor, controlled release of NO, and maintenance of ultra-low fouling property of liquid-infused materials. The LINORel tubing reduces protein adhesion as observed using fluorescence imaging, and platelet adhesion (81.7 ± 2.5%) in vitro over a 2 h period. The LINORel combination greatly reduces bacterial adhesion and biofilm formation of two most common pathogens responsible for hospital acquired infections: gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa (99.3 ± 1.9% and 88.5 ± 3.3% respectively) over a 7-day period in a CDC bioreactor environment. Overall, the LINORel approach provides a synergistic combination of active and passive non-fouling approaches to increase biocompatibility and reduce infection associated with medical devices.

Dose-dependent inhibition of uterine contractility by nitric oxide: A potential mechanism underlying persistent breeding-induced endometritis in the mare.

Nitric oxide (NO) may have a role in persistent breeding-induced endometritis in mares through an inhibitory effect on uterine contractility. The objectives of this study were to test the effect of NO on spontaneous uterine contractility in-vitro and to evaluate whether this effect varied between the longitudinal and circular muscle layers of the uterus. Reproductive tracts were collected from eight euthanized non-pregnant mares (age 4-19 years; body weight 405-530 kg). Transrectal examination of the reproductive tract was performed before euthanasia to evaluate stage of the estrous cycle and presence of any apparent abnormality. After euthanasia, one uterine tissue sample was collected for histological evaluation and four full-thickness uterine tissue strips (10-12 mm × 2 mm), two parallel to each muscle layer, were excised for in-vitro contractility evaluation. Strips were suspended in tissue chambers containing Krebs-Henseleit solution, with continuous aeration (95% O2-5% CO2; pH 7.4) at 37 °C. After equilibration, spontaneous contractility was recorded (pre-treatment) and strips excised in each direction were randomly allocated to each of two groups: 1) SNAP (S-nitroso-N-acetylpenicillamine, an NO donor); or 2) NAP (N-acetyl-d-penicillamine, vehicle and time-matched control). These were treated at 15 min intervals with increasing concentrations (10-7 M to 10-3 M) of SNAP and NAP, respectively. Contractility data was recorded throughout the experiment. An interaction effect of group-by-concentration was observed (P < 0.0001). The mean contractility after treatment with 10-4 M and 10-3 M SNAP were significantly lower than the pre-treatment contractility and the mean contractility after treatment with lower SNAP concentrations. In contrast, contractility did not change significantly in the NAP treated controls. The effect of treatment on uterine contractility was not influenced by age or weight of the mare, stage of estrous cycle, uterine histology grade, or muscle layer. Secondary findings included significant main effects of stage of estrous cycle (increased contractility in estrus compared to diestrus), uterine histology grade (decreased contractility in grade IIB compared to grade I) and age (decreased contractility in mares aged > 8 years compared to mares aged ≤ 8 years). In conclusion, results of this study indicate that NO has a dose-dependent inhibitory effect on spontaneous uterine contractility irrespective of the muscle layer in the mare.

Blunted flow-mediated responses and diminished nitric oxide synthase expression in lymphatic thoracic ducts of a rat model of metabolic syndrome.

Shear-dependent inhibition of lymphatic thoracic duct (TD) contractility is principally mediated by nitric oxide (NO). Endothelial dysfunction and poor NO bioavailability are hallmarks of vasculature dysfunction in states of insulin resistance and metabolic syndrome (MetSyn). We tested the hypothesis that flow-dependent regulation of lymphatic contractility is impaired under conditions of MetSyn. We utilized a 7-wk high-fructose-fed male Sprague-Dawley rat model of MetSyn and determined the stretch- and flow-dependent contractile responses in an isobaric ex vivo TD preparation. TD diameters were tracked and contractile parameters were determined in response to different transmural pressures, imposed flow, exogenous NO stimulation by S-nitro-N-acetylpenicillamine (SNAP), and inhibition of NO synthase (NOS) by l-nitro-arginine methyl ester (l-NAME) and the reactive oxygen species (ROS) scavenging molecule 4-hydroxy-tempo (tempol). Expression of endothelial NO synthase (eNOS) in TD was determined using Western blot. Approximately 25% of the normal flow-mediated inhibition of contraction frequency was lost in TDs isolated from MetSyn rats despite a comparable SNAP response. Inhibition of NOS with l-NAME abolished the differences in the shear-dependent contraction frequency regulation between control and MetSyn TDs, whereas tempol did not restore the flow responses in MetSyn TDs. We found a significant reduction in eNOS expression in MetSyn TDs suggesting that diminished NO production is partially responsible for impaired flow response. Thus our data provide the first evidence that MetSyn conditions diminish eNOS expression in TD endothelium, thereby affecting the flow-mediated changes in TD lymphatic function.

Orthogonal Cysteine-Penicillamine Disulfide Pairing for Directing the Oxidative Folding of Peptides.

Precise disulfide pairing in synthetic peptides usually is achieved using orthogonal protecting group strategies or relies on primary sequence manipulation. Orthogonal disulfide pairing technology should be promising for directing the rational folding of multicyclic peptides from the fully reduced peptides. Here, we report a discovery on the orthogonality between heterodisulfide pairing of cysteine (Cys) and penicillamine (Pen) and formation of Cys-Cys/Pen-Pen homodisulfides. The orthogonal Cys-Pen disulfide pairing can be exploited for highly selective production of certain (multi)cyclic structures (or even a sole structure without isomers) through direct oxidation in air or thiol-disulfide exchanges in redox media. This strategy makes rational folding of multicyclic peptides without protecting groups, sequence manipulation, and complex synthetic reactions a reality, thus providing invaluable assets to peptide communities, and should greatly benefit the development of multicyclic peptide therapeutics and ligands.

99mTc-d-penicillamine-glucuronide: synthesis, radiolabeling, in vitro and in vivo evaluation.

The current study was aimed at synthesizing a glucuronide derivative of D-penicillamine (D-PA) to be used for imaging purposes. First of all, D-PA-glucuronide (D-PA-Glu) was synthesized by experimental treatments starting with uridine 5'-diphospho-glucuronosyltransferase enzyme rich microsome preparate. Then, the synthesized compound was labeled with technetium ((99m)Tc) by using a reduction method with stannous chloride. Quality controls were performed by using high-performance liquid chromatography and thin-layer radio chromatography (TLRC). Radiolabeling yield of (99m)Tc-D-PA-Glu was more than 98% according to TLRC results. In vitro evaluations of radiolabeled complexes were investigated on PC-3 human prostate cancer cells. (99m)Tc-D-PA-Glu exhibited more accumulation on PC-3 cells versus (99m)Tc-D-PA at 240 minutes. In order to determine its radiopharmaceutical potential, biodistribution studies were carried out in male Albino Wistar rats. The biodistribution results of (99m)Tc-D-PA-Glu, showed the highest uptake in prostate at 120 minutes postinjection with the main excretion route being through kidneys and bladder. (99m)Tc-D-PA-Glu and (99m)Tc-D-PA have exhibited different biodistribution results.

N-acetylpenicillamine inhibits the replication of porcine reproductive and respiratory syndrome virus in vitro.

Nitric oxide (NO) was proposed to be an important molecule against some microorganisms. In this study, we investigated the inhibitory effect of NO on the infection by porcine reproductive and respiratory syndrome virus (PRRSV) in vitro and the role of NO in the defense against PRRSV. Our results indicated that exogenous NO did not inhibit PRRSV infection. Unexpectedly, N-acetylpenicillamine (NAP), a commonly used compound as negative control for NO-producing reagents, inhibited PRRSV replication. Thus, the inhibition effect of NAP on PRRSV replication was further explored. We found that the maximal inhibition effect of NAP on PRRSV replication was achieved upon treatment 1 h after virus infection and the virus yield was reduced by approximately 50 fold in the presence of 400 muM NAP. An obvious inhibitory effect on viral RNA and protein synthesis was also observed. However, the inhibitory effect was only achieved at early phase of virus infection. The normal virus yield could be restored upon the removal of NAP treatment. The inhibitory effect might be caused by sulfhydryl-reducing capacity and metal chelating properties of NAP. These studies suggested that (i) NO production or NO synthase (NOS) expression profiling may not be a reliable index for the immune response to PRRSV; (ii) NAP could inhibit the replication of PRRSV.

A novel water-soluble gossypol derivative increases chemotherapeutic sensitivity and promotes growth inhibition in colon cancer.

Compound 1 ((-)-gossypol) has been long known as a chemical anticancer agent. With its low water solubility and toxicity, it is not widely used as a commercial drug. To overcome these disadvantages, several novel derivatives of gossypol were designed, synthesized, and analyzed. One of the derivatives, compound 7 (6-aminopenicillanic acid sodium-gossypolone), was identified with great water solubility and anticancer property, suggested by inducing a dramatically decrease in Bcl-2 and Bcl-xL protein expression level found in vitro and growth inhibition of murine colon tumor in vivo. Furthermore, it was also recognized with less toxicity than compound 1 in vivo and significantly increased chemotherapeutic sensitivity against colon cancer in combination with traditional chemotherapeutic agent 5-fluorouracil. Therefore, it is concluded that compound 7 is superior to parent compound 1, and further preclinical studies of compound 7 is necessary for colon cancer therapy.

Biological and conformational evaluation of bifunctional compounds for opioid receptor agonists and neurokinin 1 receptor antagonists possessing two penicillamines.

Neuropathic pain states and tolerance to opioids can result from system changes in the CNS, such as up-regulation of the NK1 receptor and substance P, lead to antiopioid effects in ascending or descending pain-signaling pathways. Bifunctional compounds, possessing both the NK1 antagonist pharmacophore and the opioid agonist pharmacophore with delta-selectivity, could counteract these system changes to have significant analgesic efficacy without undesirable side effects. As a result of the introduction of cyclic and topological constraints with penicillamines, 2 (Tyr-cyclo[d-Pen-Gly-Phe-Pen]-Pro-Leu-Trp-NH-[3',5'-(CF(3))(2)-Bzl]) was found as the best bifunctional compound with effective NK1 antagonist and potent opioid agonist activities, and 1400-fold delta-selectivity over the mu-receptor. The NMR structural analysis of 2 revealed that the relative positioning of the two connected pharmacophores as well as its cyclic and topological constraints might be responsible for its excellent bifunctional activities as well as its significant delta-opioid selectivity. Together with the observed high metabolic stability, 2 could be considered as a valuable research tool and possibly a promising candidate for a novel analgesic drug.

Effect of thiol drugs on tert-butyl hydroperoxide induced luminol chemiluminescence in human erythrocytes, erythrocyte lysate, and erythrocyte membranes.

The paper investigates the effect of thiol drugs (RSH) under oxidative stress condition using luminol-enhanced chemiluminescence technique. The examinations included N-acetylcysteine (NAC), N-acetylpenicillamine (NAP), penicillamine (PEN), mesna (MES), and tiopronin (TPR). The model systems contained isolated human erythrocytes (RBC), erythrocyte lysates (LYS) or erythrocyte membranes (MEM) exposed to tert-butyl hydroperoxide (t-BuOOH). Under the influence of RSH, a bimodal character of some experimental chemiluminescence curves was observed and the kinetic solution was considered as the sum of two logistic-exponential processes. These chemiluminescence changes probably reflected two connected processes--scavenging by RSH of the t-BuOOH-induced free radicals and simultaneous generation of thiol-derived secondary free radicals. Individual differences in thiols interaction showed a multivariate set of the kinetic curve descriptors. The Principal Component Analysis (PCA) well distinguished subsets of RSH influence in systems with RBC or LYS. Generally, the action of NAC was exclusively pro-oxidant in both systems, with RBC and LYS. The behaviour of MES or NAP in these systems was also pro-oxidant but many times less prominent than NAC. Under the influence of TPR a dramatic switch in the anti-oxidant effect was observed in system with RBC to very pro-oxidant effect in LYS. The influence of PEN was analogical to TPR but very weak. This experimental model together with kinetic solution of the unique bimodal chemiluminescence curves, and PCA, supply new insights to the dual (anti- and pro-oxidant) effects of thiol drugs under oxidative stress condition.

Nitric oxide induces apoptosis in GM-CSF-treated eosinophils via caspase-6-dependent lamin and DNA fragmentation.

Asthma is characterized by accumulation of eosinophils in the lungs and delayed apoptosis may be one mechanism leading to eosinophilia. Nitric oxide (NO), present in inflamed lungs, has been shown to possess both anti- and proeosinophilic properties. We previously showed that NO induces apoptosis in the presence of survival prolonging cytokine IL-5 in human eosinophils. In the present study, we examined the intracellular mechanisms of NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF)-treated eosinophils concentrating on the role of caspases and calpains. Eosinophils were isolated from human blood and apoptosis was determined by relative DNA fragmentation assay, morphological analysis and/or Annexin-V FITC assay. We showed that NO-donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) induced apoptosis in GM-CSF-treated eosinophils. SNAP-induced DNA fragmentation was totally prevented by an inhibitor of caspase-6 (Z-VEID-FMK). Decreased levels of caspase-6 proenzyme and increased amounts of cleaved lamin A/C in SNAP-treated cells indicated activation of caspase-6. Furthermore, SNAP-induced lamin A/C and B fragmentation was totally abolished by an inhibitor of caspase-6. According to our results, caspase-6 mediates lamin and DNA fragmentation also in spontaneously dying eosinophils. Inhibitor of calpains prevented most of DNA fragmentation related to spontaneous apoptosis but had no effect in eosinophils undergoing NO-induced apoptosis. In the present study we showed that caspase-6 is essential for the executive phase involving lamin and DNA fragmentation in both NO-induced and spontaneous eosinophil apoptosis. However, differences in the involvement of calpains suggest that the intracellular signalling in NO-induced apoptosis has specific features at the level of proteases. This study demonstrates new mechanisms for NO-induced and spontaneous apoptosis in human eosinophils.

Application of (S)-N-(4-Nitrophenoxycarbonyl) phenylalanine methoxyethyl ester as a chiral derivatizing reagent for reversed-phase high-performance liquid chromatographic separation of diastereomers of amino alcohols, non-protein amino acids, and PenA.

Indirect enantioresolution of 15 primary and secondary amino group containing compounds (amino alcohols, non-protein amino acids, PenA) was done using the reagent (S)-N-(4-Nitrophenoxycarbonyl) phenylalanine methoxyethyl ester [(S)-NIFE] by reversed-phase high-performance liquid chromatography. The diastereomeric derivatives were analyzed under reversed-phase conditions using linear gradient. The detection was at 205 nm and sharp peaks were obtained. The reagent used is comparatively economic than the other derivatizing reagents. Method validation was also done.

Enantioresolution of dl-penicillamine.

Penicillamine (PenA) is a nonproteinogenic amino acid containing a thiol group. The three functional groups in penicillamine undergo characteristic chemical reactions and differ in their ability to participate in various chemical and biochemical reactions. d-penicillamine is more active pharmacologically, while the l-isomer occurs 'naturally'. This review deals with the enantioresolution of PenA both by direct and indirect methods using liquid chromatography. HPLC separation of its diastereomers prepared with different chiral derivatizing reagents (on reversed-phase columns) and separation of the derivatives prepared with achiral reagents (on chiral columns or via ligand exchange mode) has been discussed. Separation of enantiomers tagged with achiral reagent (to add a chromophore for ehanced detection) when there is no diastereomer formation has been considered separately. In addition, application of PenA and its derivatives as chiral selector for enentioresolution of certain other compounds has also been discussed.